Polycation-sensing receptor in aquatic species and methods of use thereof

ABSTRACT

Polycation-sensing receptors present in aquatic species and methods of regulating polycation-sensing receptor-mediated functions in aquatic species are described.

RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.:09/715,538, filed on Nov. 17, 2000, abandoned Jun. 26, 2003, entitled,“Polycation-Sensing Receptor in Aquatic Species and Methods of UseThereof,” by H. William Harris, et al., which is a Divisional of U.S.application Ser. No.: 09/162,021, filed on Sep. 28, 1998, now U.S. Pat.No. 6,337,391, issued Jan. 8, 2002, entitled, “Polycation-SensingReceptor in Aquatic Species and Methods of Use Thereof” by H. WilliamHarris, et al., which is a continuation-in-part of International PCTapplication No. PCT/US97/05031, entitled “Polycation-Sensing Receptor inAquatic Species and Methods of Use Thereof”, by H. William Harris, etal., filed on Mar. 27, 1997, which is a continuation-in-part ofapplication Ser. No. 08/622,738, abandoned Jul. 1, 2002, entitled“Polycation-Sensing Receptor in Fish and Methods of Use Thereof”, by H.William Harris, et al., filed Mar. 27, 1996, the teachings of which arehereby incorporated herein by reference in their entirety.

GOVERMAENT SUPPORT

This invention was made with Government support under Contract No. R01DK38874 awarded by the National Institutes of Health. The Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

It is well recognized that a stagnation or decline in production ofedible seafood, in particular, fish, by the marine fishing industry hasoccurred on a world wide basis. Since the world's population increasesby approximately 100 million each year, maintenance of the presentcaloric content of the average diet will require production of anadditional 19 million metric tons of seafood per year (United NationsFood and Agriculture Organization, The State of the World Fisheries andAquaculture, Rome, Italy (1995)). In addition, fish products arebecoming increasingly utilized in ways other than just food, forexample, production of shells and pearls. To achieve this level ofproduction, aquaculture (the cultivation of marine species) will have todouble its production in the next 15 years, and wild populations ofmarine species must be restored.

Aquatic species includes marine teleost and elasmobranch fishes, freshwater teleost fish, euryhaline fish crustations, molusks andechinoderms. Marine teleost fish live in sea water with a highosmolality of about 1,000 mosm. Freshwater teleost fish normally live inwater of less than 50 mosm. Euryhaline fish have the ability toacclimate to either of these environments. Ionic composition andosmolality of fish body fluids are maintained in these vastly differentenvironments through gill, kidney and gastrointestinal tract epithelialcell function.

A major problem in aquaculture is development of methodology to rearmarine teleost fish, such as cod, flounder and halibut, under freshwaterhatchery conditions. To date, factors critical to the acclimation andsurvival of marine species to fresh water environments, and the controlof these factors, have not been fully elucidated.

Attempts to develop such methodologies have also been complicated byproblems with feeding the maturing larval forms of these fish.Development of cod, halibut or flounder species that could be reared infresh water would be of great potential benefit in this regard. Undercontrolled fresh water conditions, developing forms of these fish couldbe raised in the absence of bacterial contamination normally present inseawater, and utilize new fresh water food sources that wouldpotentially improve their survival.

The aquaculture industry utilizes the ability of young fish, e.g.,salmon, (also called par) to be raised initially in fresh water andsubsequently to be transferred for “growth out” in salt water pens as ameans to produce large numbers of adult fish (young salmon tolerant toseawater are called smolt). Improvements in both the survival and healthof fish undergoing the par-smolt transition would be very valuable foraquaculture growers.

Moreover, salmon that are kept in coastal marine “grow-out” pens duringthe winter are constantly at risk, since both winter storms, as well asexposure to extremely cold seawater, causes fish to freeze and die.These risks are further complicated by the fact that when adult salmonare adapted to salt water they do not readily readapt back to freshwater environment. Hence, lack of understanding of the means to readaptadult salmon from salt to fresh water results in the loss of salmon.

It is apparent, therefore, that there is an immediate need to developmethods of augmenting the survival of fish in fresh water and sea water,both in a natural environment and an aquacultural environment.

SUMMARY OF THE INVENTION

The present invention relates to the identification and characterizationof a PolyValent Cation-sensing Receptor protein (also referred to hereinas the Aquatic polyvalent cation-sensing receptor, Aquatic PVCR, orPVCR) which is present in various tissues of marine species. As definedherein, aquatic species includes various fish (e.g., elasmobranch fish,such as sharks, skates; teleost fish, such as summer and winterflounder, salmon, cod, halibut, lumpfish and trout), crustaceans (e.g.,lobster, crab and shrimp), mollusks (e.g., clams, mussels and oysters),lamprey and swordfish.

As described herein, for the first time, a polyvalent cation-sensingreceptor protein has been identified in aquatic species, located on theplasma membranes of cells in the gastrointestinal tract, kidney, ovary,lung, brain and heart, and in fish brain, gill, heart, intestines,urinary bladder, rectal gland, kidney tubules, and olfactory lamellae.The widespread distribution of Aquatic PVCR protein on the plasmamembranes of epithelial cells, as well as in the brain, indicates theinvolvement of Aquatic PVCR in modulation of epithelial ion and watertransport and endocrine function. Data presented herein demonstrate thatthe Aquatic PVCR plays a critical role in the acclimation of fish toenvironments of various salinities. The Aquatic polyvalentcation-sensing receptor allows the successful adaptation of fish, suchas flounder, to marine and fresh water environments.

One embodiment of the present invention encompasses Aquatic PVCRproteins expressed in tissues of marine species. Aquatic PVCR proteinshave been identified as being present in selected epithelial cells inmarine, fresh water and euryhaline fish kidney, intestine, gill, urinarybladder, brain, and olfactory tissue. More specifically, the AquaticPVCR protein has been identified on the plasma membranes of epithelialcells of fish kidney tubules, especially in the collecting duct (CD),late distal tubule (LDT) and the olfactory lamellae. The presentinvention is intended to encompass these Aquatic PVCR proteins, theiramino acid sequences, and nucleic acid sequences, (DNA or RNA) thatencode these Aquatic PVCR proteins. In particular, the claimed inventionembodies the amino acid and nucleic acid sequences of PVCRs in dogfishshark, winter and summer flounder, and lumpfish.

In another embodiment of the present invention, methods for regulatingsalinity tolerance in fish are encompassed. Data presented hereinindicate that the Aquatic PVCR is a “master switch” for both endocrineand kidney regulation of adult fish kidney and intestinal ion and watertransport, as well as key developmental processes within the fishembryo. Modulating the expression of the Aquatic polyvalentcation-sensing receptor will activate or inhibit Aquatic PVCR mediatedion transport and endocrine changes that permit fish to adapt to freshor salt water. Also, increasing or deceasing salinity tolerance inaquatic species can refer to activating the PVCR in the epithelialcells.

For example, methods are provided to increase the salinity tolerance offish adapted to fresh water environment by activation of the AquaticPVCR in selected epithelial cells. Methods are also provided to decreasethe salinity tolerance of fish adapted to a salt water environment byinhibiting the activity of the Aquatic PVCR in selected epithelialcells. Also, regulation of salinity tolerance, via regulating theactivation/inhibition of the Aquatic PVCR, occurs by modulating the ionconcentration in the surrounding environment. Such modulation can bedone by changing the ion concentration of magnesium, calcium and/orsodium.

In another embodiment of the present invention, methods are provided toidentify a substance capable of regulating ionic composition of fishfluids, (e.g., salinity tolerance in fish), and endocrine function, bydetermining the effect that the substance has on the activation orinhibition of the Aquatic PVCR. As described herein, the nucleic acidsequence encoding an Aquatic PVCR has been determined and recombinantPVCR proteins can be expressed in e.g., oocytes of the frog, Xenopuslaevis. The oocyte assay system permits the screening of a large libraryof compounds that will either activate or inhibit Aquatic PVCR function.Candidate compounds can be further screened in e.g., an in vitro assaysystem using isolated flounder bladder preparations to measuretransepithelial transport of ions important for salinity adaption.

As a result of the work described herein, Aquatic PVCR proteins havebeen identified and their role in maintaining osmoregulation has beencharacterized. As a further result of the work described herein, methodsare now available to modulate the activation of the Aquatic PVCR,resulting in methods to regulate salinity tolerance in marine and freshwater species of fish and thus, facilitate aquaculture of marine fish.Methods of regulating salinity tolerance also provides the means todevelop new species of marine fish that are easily adaptable to freshwater aquaculture. Successful development of new species of marine fishwould permit these species to be raised initially in protected freshwater hatcheries and later transferred to marine conditions.

The claimed methods also pertain to method for altering body composition(e.g., tissue composition, or meat/muscle composition) comprisingmodulating the salinity (e.g., ion concentration) of the surroundingenvironment. Aspects of body composition that are altered include, butare not limited to: fat content, protein content, weight, thickness,moisture, and taste. For example, the thickness of a filet of fish canbe increased by the methods described herein. The altering of bodycomposition occurs by maintaining the aquatic species in low and/or highsalinity/ion concentrations.

The claimed methods also related to methods for reducing or essentiallyeliminating or ridding the fish of parasites, bacteria, andcontaminants. Maintaining aquatic species in higher salinity than normalreduces parasites and/or bacteria while maintaining the species in lowersalinity reduces contaminants (e.g., antibiotics, hydrocarbons, and/oramines). The species can be maintained in both environments,consecutively, to reduce parasites, bacteria and contaminants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A–F are photographs of immunocytochemistry results showing thedistribution of PVCR protein in various tissues of elasmobranch fish,including dogfish shark (Squalus acanthias) and little skate (Rajacrinacca).

FIGS. 2A–F are photographs of immunocytochemistry results showing thedistribution of PVCR protein in various tissues of teleost fishincluding flounder (Pseudopleuronectes americanus), trout (Onchorynchusnerka) and killifish (Fundulus heteroclitus).

FIGS. 3A–B are audioradiograms showing RNA blotting analyses.

FIGS. 4A∝E depict the nucleotide sequence of Shark Kidney CalciumReceptor Related Protein-I (SKCaR-RP-I or SKCaR-I) (SEQ ID NO: 1) withthe ORF starting at nt 439 and ending at 3516.

FIGS. 5A–E depict the annotated nucleotide sequence (SEQ ID NO: 1) andthe deduced amino acids sequence (SEQ ID NO: 2) of the Shark KidneyCalcium Receptor Related Protein-I (SKCaR-RP-I).

FIG. 6 is an autoradiogram showing the results of Northern blot analysesof A+RNA from various shark tissues.

FIGS. 7A–B are autoradiograms showing the results of RT-PCRamplifications of poly A+RNA from various aquatic species.

FIG. 8 is a photograph of immunocytochemistry results showing PVCRexpression in selected tissues of Fundulus after 18 days of exposure toeither sea or fresh water as determined by RNA blotting analysis.

FIGS. 9A–D are photographs showing the results of immunocytochemistryanalysis of PVCR expression in the kidney tubules of Fundulus fisheither chronically (18 days) or acutely (7 days) adapted to either saltor fresh water.

FIG. 10 is a graphical representation showing a normalized calciumresponse (%) against the amount of Calcium (mM) of the SKCaR-I proteinwhen modulated by alternations in extracellular NaCl concentrations.

FIG. 11 is a graphical representation showing a normalized calciumresponse (%) against the amount of magnesium(mM) of the SKCaR-I proteinin increasing amounts of extracellular NaCl concentrations.

FIG. 12 is a graphical representation showing the EC50 for calciumactivation of shark CaR (mM) against the amount of sodium (mM) of theSKCaR-I protein in increasing amounts of extracellular NaClconcentrations.

FIG. 13 is a graphical representation showing the EC50 for magnesiumactivation of shark CaR (mM) against the amount of sodium (mM) of theSKCaR-I protein in increasing amounts of extracellular NaClconcentrations.

FIG. 14 is a graphical representation showing the EC50 for magnesiumactivation of shark CaR (mM) against the amount of sodium (mM) of theSKCaR-I protein in increasing amounts of extracellular NaClconcentrations and added amounts of calcium (3mM).

FIG. 15 is a graphical representation of water transport (Jv) againstsequential exposures to Gd3+(300 μM), thiazide (100 μM) and magnesium(100 mM) and shows the response of a urinary bladder of winter flounderafter exposure of its apical membrane to various CaR agonists andhydrochlorothiazide.

FIGS. 16A–B are the nucleic acid sequence (cDNA) of a dogfish SharkCalcium Receptor Related Protein-IIa (SKCaR-IIa) (SEQ ID NO: 3).

FIG. 17 is the amino acid sequence of a dogfish Shark Calcium ReceptorRelated Protein-IIa (SKCaR-IIa) (SEQ ID NO:4).

FIGS. 18A–B is the annotated nucleic (SEQ ID NO: 3) and amino acidsequence (SEQ ID NO:4) for a dogfish Shark Calcium Receptor RelatedProtein-IIa (SKCaR-IIa).

FIG. 19 is the nucleic acid sequence (cDNA) of a dogfish Shark CalciumReceptor Related Protein-IIb (SKCaR-IIb) (SEQ ID NO: 5).

FIG. 20 is the amino acid sequence of a dogfish Shark Calcium ReceptorRelated Protein-IIb (SKCaR-IIb) (SEQ ID NO:6).

FIGS. 21A–B is the annotated nucleic (SEQ ID NO: 5) and amino acidsequence (SEQ ID NO:6) for a dogfish Shark Calcium Receptor RelatedProtein-IIb (SKCaR-IIb).

FIG. 22 is the nucleic acid sequence (cDNA) of a winter flounder (SEQ IDNO: 7) Aquatic PVCR.

FIG. 23 is the amino acid sequence of a winter flounder (SEQ ID NO:8)Aquatic PVCR.

FIGS. 24A–B is the annotated nucleic (SEQ ID NO: 7) and amino acidsequence (SEQ ID NO:8) for a winter flounder Aquatic PVCR.

FIG. 25 is the nucleic acid sequence (cDNA) of a summer flounder (SEQ IDNO: 9) Aquatic PVCR.

FIG. 26 is the amino acid sequence of a summer flounder (SEQ ID NO: 10)Aquatic PVCR.

FIG. 27 is the annotated nucleic (SEQ ID NO: 7) and amino acid sequence(SEQ ID NO:8) for a summer flounder Aquatic PVCR.

FIGS. 28A–B are the nucleic acid sequence (cDNA) of a lumpfish (SEQ IDNO: 11) Aquatic PVCR.

FIG. 29 is the amino acid sequence of a lumpfish (SEQ ID NO: 12) AquaticPVCR.

FIGS. 30A–C is the annotated nucleic (SEQ ID NO: 11) and amino acidsequence (SEQ ID NO: 12) for lumpfish Aquatic PVCR.

FIGS. 31A and B are photographs of immunochemistry of the lamellae ofthe olfactory organ epithelia of the dogfish shark using antisera 1169and a control with no antisera 1169, respectively. The darker reactionproduct indicates specific 1169 antibody binding to the apical membraneof olfactory organ epithelial cells.

DETAILED DESCRIPTION

Described herein, for the first time, are cell surface receptors, calledpolyvalent cation-sensing receptor proteins, which are present inselected epithelial cells in aquatic species tissue and organs, such asfish kidney, intestine, bladder, rectal gland, gill and brain. ThisAquatic receptor protein is also referred to herein as the “AquaticPVCR” or “PVCR.” Evidence is also presented herein that the expressionof Aquatic PVCR is modulated in aquatic species transferred from freshto salt water. The combination of these data and knowledge ofosmoregulation in fish, and other marine species, outlined brieflybelow, strongly suggest that Aquatic PVCR is the “master switch” forboth endocrine and kidney regulation of marine species kidney, intestineion and water transport. In addition, Aquatic PVCR function may controlor strongly influence maturation and developmental stages in marinespecies.

In mammals, calcium receptor protein, or terrestial CaR proteins (alsoreferred to herein as mammalian CaR) have been identified in varioustissues in humans and rat. A mammalian CaR protein has been isolated andshown to be the cell surface receptor enabling mammalian parathyroid andcalcitonin cells to respond to changes in extracellular Ca²⁺. (Brown, E.M. et al., New Eng. J. Med., 333:243, (1995)). Mammalian CaR is amembrane protein that is a member of the G-protein-coupled receptorfamily. When activated by external Ca²⁺, PVCR modulates variousintracellular signal transduction pathways and alters certain functionsin selected cells including secretion of various hormones (PTH,calcitonin, ACTH and prolactin) by endocrine/brain cells and iontransport by epithelial cells.

Subsequent work has revealed that abundant CaR/PVCR is present inepithelial cells of the thick ascending limb (TAL) and distal convolutedtubules (DCT) of the mammalian kidney where it modulates transepithelialsalt transport (Riccardi, D. J. et al., Proc. Nat. Acad. Sci USA,92:131–135 (1995)). Recent research demonstrated that PVCR is present onthe apical surface of epithelial cells of the mammalian kidney medullarycollecting duct where it senses urinary Ca²⁺ and adjustsvasopressin-mediated water reabsorption by the kidney (Sands, J. M. etal., J. Clinical Investigation 99:1399–1405 (Mar. 1997)). Lastly, PVCRis also present in various regions of the brain where it is involved inregulation of thirst and associated behavior (Brown, E. M. et al., NewEngland J. of Med., 333:234–240 (1995)).

Another protein important for osmoregulation in mammals is the NaClcotransporter. The NaCl cotransporter is present in the DCT of humankidney where it absorbs NaCl and facilitates reabsorption of Ca²⁺. ANaCl cotransporter protein has also been isolated from flounder urinarybladder (Gamba, G. et al., Proc. Nat. Acad. Sci. (USA), 902749–2753(1993)). Recently, it has been demonstrated that NaCl reabsorptionmediated by this NaCl transporter in the DCT of humans is modulated bymammalian PVCR (Plotkin, M. et al. J. Am. Soc. Nephrol., 6:349A (1995)).

As described herein, a PVCR protein has been identified in specificepithelial cells in tissues critical for ionic homeostasis in marinespecies. It is reasonable to believe that the Aquatic PVCR plays similarcritical roles in biological functions in marine species, as themammalian CaR in mammals.

Specifically, Aquatic PVCR proteins have been found in species ofelasmobranchs and species of teleosts. Elasmobranchs are cartilaginousfish, such as sharks, rays and skates, and are predominately marine;teleosts, such as summer and winter flounder, cod, trout, killifish andsalmon, can be freshwater, marine or euryhaline. The PVCR has also beenisolated several other species including lumpfish, swordfish, andlamprey.

Marine teleost fish live in seawater possessing a high osmolality (1,000mosm) that normally contains 10 millimolar (mM) Ca²⁺, 50 mM Mg²⁺ and 450mM NaCl (Evans, D. H. Osmotic and Ionic Regulation, Chapter 11 in ThePhysiology of Fishes, CRC Press, Boca Raton, Fla. (1993)). Since theirbody fluids are 300–400 mosm, these fish are obligated to drink seawater, absorb salts through their intestine and secrete large quantitiesof NaCl through their gills and Mg²⁺ and Ca²⁺ through their kidneys.Their kidneys produce only small amounts of isotonic urine.

In contrast, fresh water teleost fish possess body fluids of 300 mosmand normally live in water of less than 50 mosm containing 5–20 mM NaCland less than 1 mM Ca²⁺ and Mg²⁺. These fish drink little, but absorblarge amounts of water from their dilute environment. As a result, theirkidneys produce copious dilute urine to maintain water balance.Freshwater fish gill tissue has a low permeability to ions and gillepithelial cells extract NaCl from water (Evans, D. H., “Osmotic andIonic Regulation”, Chapter 11 in The Physiology of Fishes, CRC Press,Boca Raton, Fla. (1993)).

Euryhaline fish acclimate to various salinities by switching back andforth between these two basic patterns of ion and water transport. Forexample, when fresh water adapted teleost fish are challenged with highsalinities, their gill epithelia rapidly alter net NaCl flux such thatNaCl is secreted rather than reabsorbed (Zadunaisky, J. A. et al., Bull.MDI Biol. Lab., 32:152–156 (1992)). Reduction of extracellular Ca²⁺ from10 mM to 100 micromolar profoundly inhibits this transport process(Zadunaisky, J. A. et al., Bull. MDI Biol. Lab., 32:152–156 (1992)). Inflounder species, transfer to seawater activates a series of changes inthe kidney allowing for secretion of large quantities of Ca²⁺ and Mg²⁺by renal epithelia and recovery of water via a thiazide sensitive NaClcotransporter in the urinary bladder (Gamba, G. et al., Proc. Nat. Acad.Sci. (USA), 90-2749–2753 (1993)).

In a similar fashion, adaption of marine euryhaline fish to fresh wateris possible because of a net reversal of epithelial ionic gradients suchthat NaCl is actively reabsorbed and divalent metal ion secretion ceases(Zadunaisky, J. A. et al., Bull. MDI Biol. Lab., 32:152–156 (1992)).These changes are mediated by alterations in hormones, especiallyprolactin, cortisol and arginine vasotocin (Norris, D. O., “EndocrineRegulation of IonoOsmotic Balance in Teleosts”, Chapter 16 in VertebrateEndocrinology, Lea and Febiger, Philadelphia, Pa. (1985)). Thesealterations in a cluster of critical hormones and functional changes inepithelial transport in gill, intestine, bladder and kidney are vitalnot only to rapid euryhaline adaption, but also throughout developmentof fish embryos, larvae and during metamorphosis.

As described in detail in Example 1, Aquatic PVCR protein has beenlocalized on the plasma membrane of selected epithelial cells in marinespecies. Specifically, Aquatic PVCR has been located on the apicalmembrane of epithelial cells of the collecting duct and late distaltubule of the elasmobranch kidney. Aquatic PVCR protein has also beenfound on the apical membranes of epithelial cells in kidney tubules,gill, urinary bladder and intestine of teleosts. As used herein, theterm “apical membrane” or “apical side” refers to the “outside” of theepithelial cell exposed to e.g., urine, rather than the basal side ofthe cell exposed e.g., to the blood. The apical membrane is alsoreferred to herein as facing the lumen, or interior of e.g., the kidneytubule or intestine. Aquatic PVCR was also found in specific regions ofteleost brain.

The Aquatic PVCR has also been localize to the lamellae of the olfactoryorgan of the dogfish shark. The PVCR was located by using theseimmunochemistry methods. A detectable antibody, referred herein asantibody/antisera 1169, that is specific to a conserved region of thePVCR was used to find this PVCR. See FIG. 31 and Example 8. Aquaticspecies are able to “smell” or otherwise sense the ion concentrationsand/or salinity in their environment.

The Aquatic PVCR proteins, described herein, can be isolated andcharacterized as to its physical characteristics (e.g., molecularweight, isoelectric point) using laboratory techniques common to proteinpurification, for example, salting out, immunoprecipation, columnchromatography, high pressure liquid chromatography or electrophoresis.Aquatic PVCR proteins referred to herein as “isolated” are Aquatic PVCRproteins separated away from other proteins and cellular material oftheir source of origin. These isolated Aquatic PVCR proteins includeessentially pure protein, proteins produced by chemical synthesis, bycombinations of biological and chemical synthesis and by recombinantmethods.

Aquatic PVCR proteins can be further characterized as to its DNA andencoded amino acid sequences as follows: A complementary DNA (cDNA)encoding a highly conserved region of the mammalian CaR, as described inBrown, E. G. et al., Nature, 366:575–580 (1993) or Riccardi, D. J. etal., Proc. Nat. Acad. Sci USA, 92:131–135 (1995), the teachings of whichare incorporated by reference, can be used as a probe to screen a cDNAlibrary prepared from e.g., flounder urinary bladder cells to identifyhomologous receptor proteins. Techniques for the preparation of a cDNAlibrary are well-known to those of skill in the art. For example,techniques such as those described in Riccardi, D. J. et al., Proc. Nat.Acad. Sci USA, 15 92:131–135 (1995), the teachings of which areincorporated herein by reference, can be used. Positive clones can beisolated, subcloned and their sequences determined.

Using the sequences of either a full length or several partial cDNAs,the complete nucleotide sequence of the flounder PVCR can be obtainedand the encoded amino acid sequence deduced. The sequences of theAquatic PVCR can be compared to mammalian CaRs to determine differencesand similarities.

Similar techniques can be used to identify homologous Aquatic PVCR inother marine species. In particular, a small peptides were used to raisean antibody that is specific to PVCRs. In particular, two antisera weredeveloped. One antisera was raised to a 23-mer peptide, referred as,“4641 antisera or 4641 antibody.” A second antisera was raised against a17-mer peptide, referred to as “1169 antisera” or “1169 antibody.” Bycomparing mammalian receptors and determining a conserved region that iscommon to all, both the 23-mer and 17-mer peptide were identified andused.

The 23-mer peptide has the sequence: DDDYGRPGIEKFREEAEERDICI (SEQ IDNO.: 13). The 17-mer peptide has the sequence: ARSRNSADGRSGDDLPC.(SEQ IDNO.: 14).

Recombinant Aquatic PVCR proteins can be expressed according to methodswell-known to those of skill in the art. For example, PVCR can beexpanded in oocytes of the frog, Xenopus laevis, both to prove identityof the cDNA clone and to determine the profile of activation of AquaticPVCR proteins as compared to mammalian CaR proteins. Exemplarytechniques are described in (Brown, E. G. et al., Nature, 366:575–580(1993); Riccardi, D. J. et al., Proc. Nat. Acad. Sci USA, 92:131–135(1995)), the teachings of which are incorporated herein by reference.

As described in Example 2, a 4.4 kb homolog of the mammalian CaR hasbeen found in flounder urinary bladder together with abundant 3.8 kbthiazide-sensitive NaCl cotransporter transcript. Using a homologycloning strategy, a cDNA library from dogfish shark kidney was preparedand screened to obtain multiple cDNA clones with partial homology tomammalian CaRs as described in Example 3. One clone called SharkKidney-Calcium Receptor Related Protein (SKCaR-RP) was isolated andcharacterized. SKCAR-RP (also referred to herein as Shark Aquatic PVCR)is 4,131 nucleotides in size (SEQ ID NO: 1). As shown in FIGS. 4A–F, thecomplete nucleotide sequence of SKCAR-RP reveals that the clone iscomposed of 438 nts of 5′ untranslated region or UTR followed by asingle open reading frame (ORF) of 3,082 nts followed by 610 nts of 3′UTR containing regions of poly A+ RNA. A clone that expresses the sharkPVCR was deposited under conditions of the Budapest Treaty with theAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110–2209, USA on Jan. 28, 1998, under accession numberATCC 209602.

FIGS. 5A–E show the ORF of the SKCAR-RP in single letter amino aciddesignations (SEQ ID NO: 2). The deduced amino acid sequence of SKCAR-RPpredicts a protein of approximately 110,00 daltons that is 74%homologous to both the rat kidney PVCR protein as well as bovineparathyroid PVCR protein. As described herein, the homology wasdetermined by BLAST software. Analysis of the amino acid sequencereveals that SKCAR-RP possesses general features that are homologous toPVCR proteins including a large extracellular domain, 7 transmembranedomains and cytoplasmic carboxyl terminal domain. In this regard, manyamino acids demonstrated to be critical to PVCR function are identicalin SKCAR-RP as compared to mammalian PVCR proteins including specificregions of the extracellular domain and the 7 transmembrane domains. Incontrast, other regions are highly divergent, including the amino acidsnumber 351–395 in the extracellular domain as well as the most of thecarboxyl terminal region (e.g., amino acids 870–1027). Importantly, theregion of amino acids present in mammalian CaRs that was used togenerate anti-CaR antiserum is also present in SKCAR-RP.

As shown in FIG. 6, Northern blot analysis of MRNA from various sharktissues reveals the highest degree of SKCAR-RP in gill followed bykidney and then rectal gland. These data are highly significant sincethese tissues have been demonstrated to be involved with ion and watertransport and body homeostasis and possess epithelial cells that stainwith anti-CaR antiserum. There appears to be at least 3 distinct MRNAspecies of approximately 7 kb, 4.2 kb and 2.6 kb that hybridize toSKCAR-RP. The 4.2 kb likely corresponds to the SKCAR-RP clone describedabove.

RT-PCR amplifications were performed as described in Example 3 afterisolation of poly A+ RNA from various aquatic species. Primers thatpermit selective amplification of a region of CaRs (nts 597–981 ofRaKCaR cDNA) that is 100% conserved in all mammalian CaRs were utilizedto obtain the sequences of similar CaRs in aquatic species. Theseprimers amplify a sequence of 384 nt that is present in theextracellular domain of CaRs and presumably is involved in bindingdivalent metal ions. The resulting amplified 384 bp cDNA was ligatedinto a cloning vector and transformed into E. coli cells for growth,purification and sequencing.

As shown in FIGS. 7A and B, partial cDNA clones have been obtained from:

dogfish shark kidney (lane 2), flounder urinary bladder (lane 3),lumpfish liver (lane 5), lobster muscle (lane 8), clam gill (lane 9) andsea cucumber respiratory tissue (lane 10) using these identical primers.Some tissues (flounder brain-lane 7) did not yield a corresponding 384nt cDNA despite careful controls. Similarly, no 384 nt cDNA was obtainedwhen only water and not RT reaction mixture was added. These datasuggest these 384 nt cDNAs are specific and not expressed in all tissuesof aquatic organisms. Each of these 384 nt cDNAs was sequenced and foundto contain a conserved nucleotide sequence identical to that present inmammalian CaRs. These data suggest the presence of CaR related proteinsin classes of aquatic organisms that are widely divergent in evolution.These include teleost fish (flounder, lumpfish), elasmobranch fish(dogfish shark), crustaceans (lobster), mollusks (clam) and echinoderms(sea cucumber).

It is important to note that Aquatic PVCR sequence obtained from theseclones shared complete identity of the 384 nt segment of mammalian CaRs.However, the Aquatic PVCR sequence obtained from the shark kidney clonedid not. These data suggest that at least two different classes ofaquatic polyvalent cation-sensing receptors exist.

In fact, additional nucleic acid sequences that encodes a PVCR wereisolated from the dogfish shark. These nucleic acid sequences, SEQ IDNOs: 3 and 5, are shown in FIGS. 16 and 19, respectively. SEQ ID NO: 3is 784 nt with an open reading frame coding for 261 amino acids (SEQ IDNO: 4, FIGS. 17 and 19). SEQ ID NO: 5 is 598 nt long and encodes a 198amino acid sequence peptide (SEQ ID NO: 6, FIGS. 20 and 21). It isreasonable to believe that these proteins also sense polyvalent cations,as described herein. The annotated sequences for SEQ ID Nos: 3 and 5 canbe found in FIGS. 18A–B and 21A–B, respectively, along with the deducedamino acid sequences (SEQ ID NOs: 4 and 6). See Example 9.

PVCRs of additional aquatic species have been isolated. For example,nucleic and amino acid sequences for Winter Flounder, Summer Flounder,and Lumpfish have been identified and determined. These sequences weredetermined using methods described herein and known in the art. Thenucleic acid sequences for Winter Flounder (SEQ ID NO: 7), SummerFlounder (SEQ ID NO: 9) and for Lumpfish (SEQ ID NO: 11) can be found inFIGS. 22, 25, and 28, respectively. The corresponding deduced amino acidsequences for Winter Flounder (SEQ ID NO: 8), Summer Flounder (SEQ IDNO: 10) and for Lumpfish (SEQ ID NO: 12) can be found in FIGS. 23, 26,and 29, respectively. See Example 9. Clones, containing sequences forWinter Flounder, Summer Flounder, and Lumpfish were deposited under theBudapest Treaty with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110–2209, USA on Oct. 5, 2000under accession numbers PTA-2545, PTA-2540, and PTA-2540, respectively.

Additionally, the nucleic and amino acid sequences for an aquatic PVCRwere isolated in Swordfish and Lamprey. These sequences were isolated asdescribed herein. These PVCR's function similar to the shark PVCR, asdescribed herein and is capable of sensing ion concentrations/salinity.

The present invention is intended to encompass Aquatic PVCR proteins,and proteins and polypeptides having amino acid sequences analogous tothe amino acid sequences of Aquatic PVCR proteins. Such polypeptides aredefined herein as Aquatic PVCR analogs (e.g., homologues), or mutants orderivatives. Analogous amino acid sequences are defined herein to meanamino acid sequences with sufficient identity of Aquatic PVCR amino acidsequence to possess the biological activity of an Aquatic PVCR. Forexample, an analog polypeptide can be produced with “silent” changes inthe amino acid sequence wherein one, or more, amino acid residues differfrom the amino acid residues of the Aquatic PVCR protein, yet stillpossesses the biological activity of Aquatic PVCR. Examples of suchdifferences include additions, deletions or substitutions of residues ofthe amino acid sequence of Aquatic PVCR.

Also encompassed by the present invention are analogous polypeptidesthat exhibit greater, or lesser, biological activity of the Aquatic PVCRproteins of the present invention.

The claimed Aquatic PVCR protein and nucleic acid sequence includehomologues, as defined herein. The homologous proteins and nucleic acidsequences can be determined using methods known to those of skill in theart. Initial homology searches can be performed at NCBI against theGenBank (release 87.0), EMBL (release 39.0), and SwissProt (release30.0) databases using the BLAST network service. Altshul, S F, et al,Basic Local Alignment Search Tool, J. Mol. Biol. 215: 403 (1990), theteachings of which are incorporated herein by reference. Computeranalysis of nucleotide sequences can be performed using the MOTIFS andthe FindPatterns subroutines of the Genetics Computing Group (GCG,version 8.0) software. Protein and/or nucleotide comparisons can also beperformed according to Higgins and Sharp (Higgins, D. G. and P. M.Sharp, “Description of the method used in CLUSTAL,” Gene, 73: 237–244(1988)). Homologous proteins and/or nucleic acid sequences to the PVCRprotein and/or nucleic acid sequences that encode the PVCR protein aredefined as those molecules with greater than 70% sequences identityand/or similarity (e.g., 75%, 80%, 85%, 90%, or 95% homology).

The “biological activity” of Aquatic PVCR proteins is defined herein tomean the osmoregulatory activity of Aquatic PVCR mammalian PVCR proteinshave been shown to mediate physiological responses to changes in bodyosmolality and salt content in kidney, parathyroid, calcitonin and braincells. (Brown, E. M. et al., New Eng. J. Med., 333:243, (1995);Riccardi, D. J. et al., Proc. Nat. Acad. Sci USA, 92:131–135 (1995);Sands, J. M. etal., Nature (Medicine) (1995); Brown, E. M. et al., NewEngland J. of Med., 333:234–240 (1995)). It is reasonable to believethat Aquatic PVCR proteins will possess identical, or similarosmoregulatory activities as these previously identified mammalian CaRproteins in fish kidney, gill, bladder, intestine, rectal gland andbrain cells. Assay techniques to evaluate the biological activity ofAquatic PVCR proteins and their analogs are described in Brown, E. M. etal., New Eng. J. Med., 333:243, (1995); Riccardi, D. J. et al., Proc.Nat. Acad. Sci USA, 92:131–135 (1995); Sands, J. M. et al., Nature(Medicine) (1995); Brown, E. M. et al., New England J. of Med.,333:234–240 (1995), the teachings of which are incorporated herein byreference. Additional assays to evaluate biological activity of PVCRproteins are described in U.S. Serial No. 60/003,697, the teachings ofwhich are also incorporated herein, in its entirety, by reference.

The “biological activity” of Aquatic PVCR is also defined herein to meanthe ability of the Aquatic PVCR to modulate signal transduction pathwaysin specific marine species cells. In mammals, studies in normal tissues,in oocytes using recombinantly expressed CaR, and cultured cells havedemonstrated that mammalian CaR protein is capable of complexing with atleast two distinct types of GTP-binding (G) proteins that transmit theactivation of CaR by an increase in extracellular calcium to variousintracellular signal transduction pathways. One pathway consists ofmammalian CaR coupling with an inhibitory Gi protein that, in turn,couples with adenylate cyclase to reduce intracellular CAMPconcentrations. A second distinct pathway consists of CaR coupling tostimulatory Gq/Gall G protein that couples with phospholipase C togenerate inositol 1,4,5 triphosphosphate that, in turn, stimulates bothprotein kinase C activity and increases intracellular Ca²⁺concentrations. Thus, depending on the distribution and nature ofvarious signal transduction pathway proteins that are expressed incells, biologically active mammalian CaRs modulate cellular functions ineither an inhibitory or stimulatory manner. It is reasonable to believethat biologically active Aquatic PVCR possesses similar signaltransduction activity.

The term “biologically active” also refers to the ability of the PVCR tosense ion concentrations in the surrounding environment. The PVCR sensesvarious polyvalent cations including calcium, magnesium and/or sodium.The PVCR is modulated by varying ion concentrations. For instance, thePVCR may be modulated (e.g., increased expression, decreased expressionand/or activation) in response to a change (e.g., increase or decrease)in ion concentration (e.g., calcium, magnesium, or sodium). See Example6. Responses to changes in ion concentrations of a fish containing aPVCR include the ability for a fish to adapt to the changing ionconcentration. Such responses include the amount the fish drinks, theamount of urine output, and the amount of water absorption. Responsesalso include changes biological processes that affect the bodycomposition of the fish and its ability to excrete contaminants.

The claimed PVCR proteins also encompasses biologically activepolypeptide fragments of the Aquatic PVCR proteins, described herein.Such fragments can include only a part of the full-length amino acidsequence of an Aquatic PVCR yet possess osmoregulatory activity. Forexample, polypeptide fragments comprising deletion mutants of theAquatic PVCR proteins can be designed and expressed by well-knownlaboratory methods. Such polypeptide fragments can be evaluated forbiological activity, as described herein.

Antibodies can be raised to the Aquatic PVCR proteins and analogs, usingtechniques known to those of skill in the art. These antibodiespolyclonal, monoclonal, chimeric, or fragments thereof, can be used toimmunoaffinity purify or identify Aquatic PVCR proteins contained in amixture of proteins, using techniques well known to those of skill inthe art. These antibodies, or antibody fragments, can also be used todetect the presence of Aquatic PVCR proteins and homologs in othertissues using standard immunochemistry methods.

The present invention also encompasses isolated nucleic acid sequencesencoding the Aquatic PVCR proteins described herein, and fragments ofnucleic acid sequences encoding biologically active PVCR proteins.Fragments of the nucleic acid sequences, described herein, as useful asprobes to detect the presence of marine species CaR. Specificallyprovided for in the present invention are DNA/RNA sequences encodingAquatic PVCR proteins, the fully complementary strands of thesesequences, and allelic variations thereof. Also encompassed by thepresent invention are nucleic acid sequences, genomic DNA, cDNA, RNA ora combination thereof, which are substantially complementary to the DNAsequences encoding Aquatic PVCR, and which specifically hybridize withthe Aquatic PVCR DNA sequences under conditions of stringency known tothose of skill in the art, those conditions being sufficient to identifyDNA sequences with substantial nucleic acid identity. As defined herein,substantially complementary means that the sequence need not reflect theexact sequence of Aquatic PVCR DNA, but must be sufficiently similar inidentity of sequence to hybridize with Aquatic PVCR DNA under stringentconditions.

Conditions of stringency are described in e.g., Ausebel, F. M., et al.,Current Protocols in Molecular Biology, (Current Protocols, 1994). Forexample, non-complementary bases can be interspersed in the sequence, orthe sequences can be longer or shorter than Aquatic PVCR DNA, providedthat the sequence has a sufficient number of bases complementary toAquatic PVCR to hybridize therewith. Exemplary hybridization conditionsare described herein and in Brown, E. M., et al. Nature, 366:575 (1993).For example, conditions such as 1×SSC 0. 1% SDS, 50°, or 0.5×SSC, 0. 1%SDS, 50° can be used as described in Examples 2 and 3.

The Aquatic PVCR DNA sequence, or a fragment thereof, can be used as aprobe to isolate additional Aquatic PVCR homologs. For example, a cDNAor genomic DNA library from the appropriate organism can be screenedwith labeled Aquatic PVCR DNA to identify homologous genes as describedin e.g., Ausebel, F. M., et al., Current Protocols in Molecular Biology,(Current Protocols, 1994).

Typically the nucleic acid probe comprises a nucleic acid sequence (e.g.SEQ ID NO: 1, 3, 5, 7, 9, or 11) and is of sufficient length andcomplementarity to specifically hybridize to nucleic acid sequenceswhich encode Aquatic species PVCR.

The requirements of sufficient length and complementarity can be easilydetermined by one of skill in the art.

Uses of nucleic acids encoding cloned receptors or receptor fragmentsinclude one or more the following: (1) producing receptor proteins whichcan be used, for example, for structure determination, to assay amolecule's activity on a receptor, and to obtain antibodies binding tothe receptor; (2) being sequenced to determine a receptor's nucleotidesequence which can be used, for example, as a basis for comparison withother receptors to determine conserved regions, determine uniquenucleotide sequences for normal and altered receptors, and to determinenucleotide sequences to be used as target sites for antisense nucleicacids, ribozymes, hybridization detection probes, or PCR amplificationprimers; (3) as hybridization detection probes to detect the presence ofa native receptor and/or a related receptor in a sample; and (4) as PCRprimers to generate particular nucleic acid sequence regions, forexample to generate regions to be probed by hybridization detectionprobes.

The claimed PVCR proteins and/or nucleic acid sequences include fragmentthereof. Preferably, the nucleic acid contains at least 14, at least 20,at least 27, at least 45, and at least 69, contiguous nucleic acids of asequence provided in SEQ. ID. NO. 1, SEQ. ID. NO. 3, SEQ. ID. NO. 5,SEQ. ID. NO. 7, SEQ. ID. NO. 9, or SEQ. ID. NO. 11. Advantages oflonger-length nucleic acid include producing longer-length proteinfragments having the sequence of a calcium receptor which can be used,for example, to produce antibodies; increased nucleic acid probespecificity under high stringent hybridization assay conditions; andmore specificity for related inorganic ion receptor nucleic acid underlower stringency hybridization assay conditions.

Another aspect of the present invention features a purified nucleic acidencoding an inorganic ion receptor or fragment thereof. The nucleic acidencodes at least 6 contiguous amino acids provided in SEQ. ID. NO. 2,SEQ. ID. NO. 4, SEQ. ID. NO. 6, SEQ. ID. NO. 8, SEQ. ID. NO. 10, SEQ.ID. NO. 12, or SEQ ID NO: 14. Due to the degeneracy of the genetic code,different combinations of nucleotides can code for the same polypeptide.Thus, numerous inorganic ion receptors and receptor fragments having thesame amino acid sequences can be encoded for by difference nucleic acidsequences. In preferred embodiments, the nucleic acid encodes at least12, at least 18, at least 23, or at least 54 contiguous amino acids ofSEQ. ID. NO. 2, SEQ. ID. NO. 4, SEQ. ID. NO. 6, SEQ. ID. NO. 8, SEQ. ID.NO. 10, SEQ. ID. NO. 12, or SEQ ID NO: 14.

Another aspect of the present invention features a purified nucleic acidhaving a nucleic acid sequence region of at least 12 contiguousnucleotides substantially complementary to a sequence region in SEQ. ID.NO. 1, SEQ. ID. NO. 3, SEQ. ID. NO. 5, SEQ. ID. NO. 7, SEQ. ID. NO. 9,or SEQ. ID. NO. 11. By “substantially complementary” is meant that thepurified nucleic acid can hybridize to the complementary sequence regionin nucleic acid encoded by SEQ. ID. NO. 1, SEQ. ID. NO. 3, SEQ. ID. NO.5, SEQ. ID. NO. 7, SEQ. ID. NO. 9, or SEQ. ID. NO. 11 under stringenthybridizing conditions. Such nucleic acid sequences are particularlyuseful as hybridization conditions, only highly complementary nucleicacid sequences hybridize. Preferably, such conditions preventhybridization of nucleic acids having 4 mismatches out of 20 contiguousnucleotides, more preferably 2 mismatches out of 20 contiguousnucleotides, most preferably one mismatch out of 20 contiguousnucleotides. In preferred embodiments, the nucleic acid is substantiallycomplementary to at least 20, at least 27, at least 45, or at least 69contiguous nucleotides provided in SEQ. ID. NO. 1, SEQ. ID. NO. 3, SEQ.ID. NO. 5, SEQ. ID. NO. 7, SEQ. ID. NO. 9, or SEQ. ID. NO. 11.

Another aspect of the present invention features a purified polypeptidehaving at least 6 contiguous amino acids of an amino acid sequenceprovided in SEQ. ID. NO. 2, SEQ. ID. NO. 4, SEQ. ID. NO. 6, SEQ. ID. NO.8, SEQ. ID. NO. 10, or SEQ. ID. NO. 12. By “purified” in reference to apolypeptide is meant that the polypeptide is in a form (i.e., itsassociation with other molecules) distinct from naturally occurringpolypeptide. Preferably, the polypeptide is provided as substantiallypurified preparation representing at least 75 %, more preferably 85 %,most preferably 95 % or the total protein in the preparation. Inpreferred embodiments, the purified polypeptide has at least 12, 18, 23,or 54 contiguous amino acids of SEQ. ID. NO. 2, SEQ. ID. NO. 4, SEQ. ID.NO. 6, SEQ. ID. NO. 8, SEQ. ID. NO. 10, or SEQ. ID. NO. 12.

Preferred receptor fragments include those having functional receptoractivity, a binding site, epitope for antibody recognition (typically atleast six amino acids) (e.g., antisera 1169). Such receptor fragmentshave various uses such as being used to obtain antibodies to aparticular region and being used to form chimeric receptors withfragments of other receptors create a new receptor having uniqueproperties.

The invention also features derivatives of full-length inorganic ionreceptors and fragments thereof having the same, or substantially thesame, activity as the full-length receptor or fragment. Such derivativesinclude amino acid addition(s), substitution(s), and deletion(s) to thereceptor which do not prevent the derivative receptor from carrying outone or more of the activities of the parent receptor.

Another aspect of the present invention features a recombinant cell ortissue. The recombinant cell or tissue is made up of a recombinednucleic acid sequence encoding at least 6 contiguous amino acidsprovided in SEQ. ID. NO. 2, SEQ. ID. NO. 4, SEQ. ID. NO. 6, SEQ. ID. NO.8, SEQ. ID. NO. 10, or SEQ. ID. NO. 12 and a cell able to express thenucleic acid. Recombinant cells have various uses including acting asbiological factories to produce polypeptides encoded for by therecombinant nucleic acid, and for producing cells containing afunctioning PVCR protein. Cells containing a functioning PVCR can beused, for example, to screen to antagonists or agonists.

As described in Example 4, it is demonstrated that the Aquatic PVCRprotein plays a critical role in the adaption of euryhaline fish toenvironments of various salinities. Adaption of the killifish, Fundulusheteroculitus, to seawater resulted in steady state expression ofAquatic PVCR MRNA in various tissues.

It is also demonstrated herein that PVCR protein undergoes rearrangementwithin epithelial cells of the urinary bladder in flounder adapted tobrackish water as compared to full strength sea water. This directlycorrelates with alterations the rate of NaCl transport by these cells.

Preliminary experiments shows that winter flounder were adapted to livein 1/10th seawater (100 mOsm/kg) by reduction in salinity from 450 mMNaCl to 45 mM NaCl over an interval of 8 hrs. (Further experimentationillustrated that winter and summer flounder can be maintained in 1/10 ortwice the salinity for over a period of 6 months.) After a 10 dayinterval where these fish were fed a normal diet, the distribution ofthe PVCR in their urinary bladder epithelial cells was examined usingimmunocytochemistry. PVCR immunostaining is reduced and localizedprimarily to the apical membrane of epithelial cells in the urinarybladder. In contrast, the distribution of PVCR in epithelial cellslining the urinary bladders of control flounders continuously exposed tofull strength seawater is more abundant and present in both the apicalmembranes as well as in punctate regions throughout the cell. These dataare consistent with previous Northern data since more PVCR protein ispresent in the urinary bladders of seawater fish vs fish adapted tobrackish water. These data suggest that PVCR protein may be present invesicles in epithelial cells of the urinary bladder and that in responseto alterations in salinity, these vesicles move from the cell cytoplasmto the apical surface of these epithelial cells. Since these sameepithelial cells possess abundant NaCl cotransporter protein that isresponsible for water reabsorption in the urinary bladder, these datasuggest that the PVCR protein modulates NaCl transport in the flounderurinary bladder by altering the proportion of NaCl cotransporter proteinthat is present in the apical membrane. As urinary Mg²⁺ and Ca²⁺concentrations increase when fish are present in full strength seawater, activation of apical PVCR protein causes endocytosis and removalof NaCl cotransporter from the apical membrane and thus reduction inurinary bladder water transport.

As a result of the work described herein, methods are now provided thatfacilitate euryhaline adaptation of fish to occur, and improve theadaption. More specifically, methods are now available to regulatesalinity tolerance in fish by modulating (e.g., alternating, activatingand or expressing) the activity of the Aquatic PVCR protein present inepithelial cells involved in ion transport, as well as in endocrine andnervous tissue. For example, salinity tolerance of fish adapted (oracclimated) to fresh water can be increased by activating the AquaticPVCR, for example, by increasing the expression of Aquatic PVCR inselected epithelial cells, resulting in the secretion of ions andseawater adaption. Specifically, this would involve regulatory eventscontrolling the conversion of epithelial cells of the gill, intestineand kidney. In the kidney, PVCR activation will facilitate excretion ofdivalent metal ions including Ca2+ and Mg²⁺ by renal tubules. In thegill, PVCR activation will reduce reabsorption of ions by gill cellsthat occurs in fresh water and promote the net excretion of ions by gillepithelia that occurs in salt water. In the intestine, PVCR activationwill permit reabsorption of water and ions across the G.I. tract aftertheir ingestion by fish.

Alternatively, the salinity tolerance of fish adapted to seawater can bedecreased by inhibiting the Aquatic PVCR, for example, by decreasing theexpression of Aquatic PVCR in selected epithelial cells, resulting inalterations in the absorption of ions and freshwater adaption. Selectedepithelial cells include, e.g., kidney, bladder, intestinal and gillcells.

The presence of Aquatic PVCR in brain reflects both its involvement inbasic neurotransmitter release via synaptic vesicles (Brown, E. M. etal., New England J. of Med., 333:234–240 (1995)), as well as itsactivity to trigger various hormonal and behavioral changes that arenecessary for adaptation to either fresh water or marine environments.For example, increases in water ingestion by fish upon exposure to saltwater is mediated by PVCR activation in a manner similar to thatdescribed for humans where PVCR activation by hypercalcemia in thesubfornical organ of the brain cause an increase in water drinkingbehavior (Brown, E. M. et al., New England J. of Med., 333:234–240(1995)). In fish, processes involving both alterations in serum hormonallevels and behavioral changes are mediated by the brain. These includethe reproductive and spawning of euryhaline fish in fresh water aftertheir migration from salt water as well as detection of salinity oftheir environment for purposes of feeding, nesting, migration andspawning.

Data obtained recently from mammals now suggest that PVCR activationplays a pivotal role in coordinating these events. For example,alterations in plasma cortisol have been demonstrated to be critical forchanges in ion transport necessary for adaptation of salmon smolts fromfresh water to salt water (Veillette, P. A., et al., Gen. and Comp.Physiol., 97:250–258 (1995). As demonstrated recently in humans, plasmaAdrenocorticotrophic Hormone (ACTH) levels that regulate plasma cortisollevels are altered by PVCR activation.

“Salinity” refers to the concentration of various ions in a surroundingaquatic environment. In particular, salinity refers to the ionicconcentration of calcium, magnesium and/or sodium (e.g., sodiumchloride). “Normal salinity” levels refers to the range of ionicconcentrations of typical water environment in which an aquatic speciesnaturally lives. For winter and summer flounder, normal salinity ornormal seawater concentrations are about 10 mM Ca, 25 mM Mg, and 450 mMNaCl. “Salinity tolerance” refers to the ability of a fish to live orsurvive in a salinity environment that is different than the salinity ofits natural environment. The upper or lower limit of ionicconcentrations in which the fish can survive have been defined. Salinitytolerance of a fish has been defined to be between at least 4× and 1/50,or 3× and 1/25, or preferably, twice and 1/10 the normal salinity.

Winter and summer flounder were maintained in at least twice the normalsalinity or 1/10 the normal salinity. See Example 10. These fish can bemaintained in these environments for long periods of time (e.g., over 3months, over 6 months, or over 1 year). These limits were defined bydecreasing or increasing the ionic concentrations of calcium, magnesium,and sodium, keeping a constant ratio between the ions. These salinitylimits can be further defined by increasing and/or decreasing anindividual ion concentration, thereby changing the ionic concentrationratio among the ions. Increasing and/or decreasing individual ionconcentrations can increase and/or decrease salinity tolerance.“Hypersalinity” or “above normal salinity” levels refers to a level ofat least one ion concentration that is above the level found in normalsalinity. “Hyposalinity” or “below normal salinity” levels refers to alevel of at least one ion concentration that is below the level found innormal salinity.

Maintaining winter and summer founder in this environment for about 3months induced noticeable and significant changes occurred to the bodycomposition of the flounder. These fish were slowly adapted to thehypersalinity or hyposalinity environments over a period of 15 days.Body composition refers to various characteristics of the fish,including, but not limited to, weight, muscle, fat, protein, moisture,taste, or thickness. Alteration of the body composition means inducing achange in one of these characteristics. Maintaining fish in 1/10 thenormal salinity results in a fish that is twice as thick, 70% fatter,and “less fishy,”(e.g., milder flavor) tasting fish than those fishmaintained in hypersalinity environments. See Example 10. A fishmaintained in low salinity or hyposalinity can increase its fat contentby at least 10% or 20%, and preferably by at least 30%, 40%, or 50% thanthose fish maintained in normal salinity. Similarly, a fish maintainedin low salinity or hyposalinity can increase its thickness by at least30% or 40%, and preferably by at least 50%, 60%, or 70% than those fishmaintained in normal salinity. A fish maintained in high salinity orhypersalinity can decrease its fat content by at least 10% or 20%, andpreferably by at least 30%, 40%, or 50% than those fish maintained innormal salinity. Similarly, a fish maintained in high salinity orhypersalinity can decrease its thickness by at least 30% or 40%, andpreferably by at least 50%, 60%, or 70% than those fish maintained innormal salinity.

Maintaining fish in a hypersalinity environment also results in fishwith a reduced number of parasites or bacteria. Preferably, theparasites and/or bacteria are reduced to a level that is safe for humanconsumption, raw or cooked. More preferably, the parasites and/orbacteria are reduced to having essentially no parasites and fewbacteria. These fish must be maintained in a hypersalinity environmentlong enough to rid the fish of these parasites or bacteria, (e.g., forat least a few days or at least a few weeks).

The host range of many parasites is limited by exposure to watersalinity. For example, Diphyllobothrium species commonly known as fishtapeworms, is encountered in the flesh of fish, primarily fresh water oreuryhaline species including flounder of salmon. Foodborne PathogenicMicroorganisms and Natural Toxins Handbook. 1991. US Food and DrugAdministration Center for Food Safety and Applied Nutrition, theteachings of which are incorporated herein by reference in theirentirety. In contrast, its presence in the flesh of completely marinespecies is much reduced or absent. Since summer flounder can survive andthrive at salinity extremes as high as 58 ppt (1.8 times normalseawater) for extended periods in recycling water, exposure of summerflounder to hypersalinity conditions might be used as a “biological”remediation process to ensure that no Diphyllobothrium species arepresent in the GI tract of summer flounder prior to their sale asproduct.

Recent data from Cole et al, J. Biol. Chem. 272:12008–12013, 1997, (theteachings of which are incorporated herein by reference in theirentirety) show that winter flounder elaborate an antimicrobial peptidefrom their skin to prevent bacterial infections. Their data reveals thatin the absence of pleurocidin, E. coli are killed by high concentrationsof NaCl. In contrast, low concentrations of NaCl (<300 mM NaCI) allow E.Coli to grow and under these conditions pleurocidin presumably helps tokill them. These data provide evidence of NaCl killing of E. Coli, aswell as highlight possible utility of bacterial elimination in fish.

Similarly, maintaining fish in a hyposalinity environment results in afish with a reduced amount of contaminants (e.g., hydrocarbons, aminesor antibiotics). Preferably, the contaminants are reduced to a levelthat is safe for human consumption, raw or cooked, and produces amilder, “less fishy” tasting fish. More preferable, the contaminants arereduced to having essentially very little contaminants left in the fish.These fish must be maintained in a hyposalinity environment long enoughto rid the fish of these contaminants, (e.g., for at least a few days ora few weeks).

Organic amines, such as trimethylamine oxide (TMAO) produce a “fishy”taste in seafood. They are excreted via the kidney in flounder. (Krogh,A. Osmotic Regulation in Aquatic Animals, Cambridge University Press,Cambridge, U.K. pgs 1–233, 1939, the teachings of which are incorporatedherein by reference in their entirety). TMAO is synthesized by marineorganisms consumed by fish that accumulate the TMAO inn their tissues.Depending on the species of fish, the muscle content of TMAO and organicamines is either large accounting for the “strong” taste of bluefish andherring or small such as in milder tasting flounder.

TMAO is an intracellular osmolyte and its accumulation in cells preventsosmotic loss of water produces by hypertonic seawater (Forster, R P andL Goldstein, Formation of Excretory Products Chapter 5 in FishPhysiology, Edited by W S Hoar, D J Randall and J R Brett Volume VIIIBioenergics and Growth. Academic Press, New York, N.Y. pages 313–345,1969, the teachings of which are incorporated herein by reference intheir entirety). The excretion of TMAO by marine teleost fish such aswinter flounder occurs almost exclusively via the kidney. Thus, in lowsalinities urine flow in winter flounder is high and dietary aminesincluding TMAO are almost completely excreted. Elger, E. B. Elger, H.Hentschel and H. Stolte, Adaptation of renal function to hypotonicmedium in the winter flounder (Psuedupleuronecies americanus). J. Comp.Physio. B157:21–30 (1987), the teachings of which are incorporatedherein in their entirety. In full strength or hyperosmotic seawater,urine flow is much diminished and amine excretion is greatly reduced andtherefore accumulates in the flounder muscle. Thus, muscle levels ofamines can be altered by subjecting flounder to differing osmoticenvironments and likely result in winter flounders with differingtastes.

Exposure of either winter or summer flounders to waters of extremedifferences in salinity (3–4 vs 58 ppt) produces profound changes in thekidney function of these fish that allow toxic compounds such asantibiotics and heavy metal to be excreted. At low salinities (3–4 ppt)the glomerular filtration and urinary flow rates are 10–100 fold largeras compared to identical fish exposed to full strength seawater. Highglomerular filtration and urine flow rates provide for a large increasein the clearance of a variety of organic compounds including antibioticsused in aquaculture (Physicians Desk Reference, 49th Edition, MedicalEconomics Data Production Company, Montvale, N.Y. page 2103, theteachings of which are incorporated herein by reference in theirentirety), as well as heavy metals including Ni²⁺, Pb²⁺ (Forster, R Pand L Goldstin. 1969. Formation of Excretory Products Chapter 5 in FishPhysiology, Edited by W S Hoar, D J Randall and J R Brett Volume VIIIBioenergics and Growth. Academic Press. New Your, N.Y. pages 313–345(the teachings of which are incorporated herein by reference in theirentirety)). Exposure of flounder to an interval of low salinity prior tomarket would produce high urine flow rates and, therefore, reduce anytissue burdens of toxic or antibiotic compounds acquired during growth.This method serves as a effective strategy to reduce environmentalcontaminants to their lowest levels possible.

Methods encompassed by the present invention include methods ofactivating or deactivating the Aquatic PVCRs described herein. The term“activation” as used herein means to make biologically functional, e.g.,rendering a cell surface receptor capable of stimulating a secondmessenger which results in modulation of ion secretion. This could be inthe form of either an inhibition of signal transduction pathways, e.g.,via a Gi protein, or stimulation of other pathways via. e.g., a Gq/Gallprotein. As a result of these alterations, ion transport by epithelialcells is reduced or stimulated. Also, activation can be related toexpression (e.g., an increase in expression).

For example, a compound, or substance, which acts as an agonist caninteract with, or bind to, the Aquatic PVCR, thereby activating theAquatic PVCR, resulting in an increase of ion secretion in selectedepithelial cells. An agonist can be any substance, or compound, thatinteracts with, or binds to, the Aquatic PVCR resulting in activation ofAquatic PVCR. Agonists encompassed by the present invention includeinorganic ions, such as the polyvalent cations calcium, magnesium andgadolinium, and organic molecules such as neomycin. Other agonists,include inorganic compounds, nucleic acids or proteins can be determinedusing the techniques described herein.

Agonists also encompassed by the present invention can include proteinsor peptides or antibodies that bind to the Aquatic PVCR resulting in itsactivation.

Activation of the Aquatic PVCR is typically direct activation. Forexample, an inorganic molecule or peptide binds directly to the receptorprotein resulting in the activation of Aquatic PVCR. However, activationof the Aquatic PVCR can also be indirect activation, such as would occurwhen e.g., an antibody is available to bind an Aquatic PVCR antagonist,thus permitting activation of the Aquatic PVCR

The term “deactivation” or “inactivation” as used herein means tocompletely inhibit or decrease biological function. For example,deactivation is when a cell surface receptor is incapable of stimulatinga second messenger. Specifically, as used herein, deactivation of theAquatic PVCR occurs when the Aquatic PVCR is rendered incapable ofcoupling with, or stimulating, a second messenger, resulting in theabsorption of ions in selected epithelial cells. Deactivation can bedirect or indirect. For example, an antagonist can interact with, orbind directly to the Aquatic PVCR, thereby rendering the Aquatic PVCRincapable of stimulation of a messenger protein.

Alternatively, deactivation can be indirect. For example, an antagonistcan deactivate Aquatic PVCR by preventing, or inhibiting an agonist frominteracting with the Aquatic PVCR. For example, a chelator can bindcalcium ions and, thus prevent the calcium ions from binding to theAquatic PVCR. Antagonists of the Aquatic PVCR can be any substancecapable of directly interacting with, or binding to, the Aquatic PVCR orinteracting with, or binding to, an agonist of the Aquatic PVCR thatresults in deactivation of the Aquatic PVCR. Antagonists encompassed bythe present invention can include, for example, inorganic molecules,organic molecules, proteins or peptides. Antagonists can also be nucleicacids, such as anti-sense DNA or RNA sequences that bind to the DNAencoding the Aquatic PVCR, thereby preventing or inhibitingtranscription into MRNA. Antagonists can also be anti-sense RNA thatbinds to the PVCR transcript, thereby preventing, or inhibitingtranslation.

Candidate substances, (e.g., compounds, peptldes or nucleic acids) to beevaluated for their ability to regulate Aquatic PVCR activity can bescreened in assay systems to determine activity. For example, one assaysystem that can be used is the frog oocyte system expressing AquaticPVCR described in Brown, E. G. et al., Nature, 366:575–580 (1993);Riccardi, D. J. et al., Proc. Nat. Acad. Sci USA, 92:131–135 (1995).

A functional assay to screen for compounds that alter PVCR mediated NaCltransport function in adult flounder urinary bladder can also be used toscreen candidate compounds for their ability to modulate Aquatic PVCR.Transport of NaCl via the thiazide sensitive NaCl cotransporter in theflounder urinary bladder is important in its adaptation to varioussalinities. NaCl transport is readily quantified using a isolatedbladder preparation from adult flounder and measurement oftransepithelial Ca²⁺ sensitive short circuit current, as described in(Gamba, G. et al., Proc. Nat. Acad. Sci. (USA), 90-2749–2753 (1993)).Use of this isolated in vitro assay system can establish a direct effectof Aquatic PVCR function or transepithelial transport of ions importantfor salinity adaptation. Compounds identified using the frog oocyteassay and in vitro NaCl transport assay system can be further tested inwhole animal adaptation experiments.

For example, to screen for PVCR reactive compounds (both agonists andantagonists) an assay previously used for study of ion and watertransport in isolated flounder urinary bladders (Renfro, L. J. Am. J.Physiol. 228:52–61, 1975) has been used. As described herein (Example5), this assay has now been adapted to screen PVCR agonists and provideddata showing that water reabsorption is >85% inhibited by application ofthiazide (specific inhibitor of the thiazide sensitive NaClcotransporter); water reabsorption is >90% inhibited by application ofgadolinium (a PVCR specific agonist); water reabsorption is >50%inhibited by application of neomycin (a PVCR specific agonist); andexposure of the bladder to PVCR agonists is reversible upon removal ofeither gadolinium or neomycin.

As a further result of the work, methods are provided to test thefunction of PVCR in developing fish, and to specifically select for fishwith altered PVCR functional and osmotic tolerance. The developmentalexpression of PVCR in developing embryo, larval and metamorphic forms offish can be determined using antibodies that recognize Aquatic PVCRand/or mammalian CaR, or by using Aquatic and/or mammalian cDNA probes,or a combination of these techniques. Initial screening of gametes,larval or metamorphic forms of fish can be tested usingimmunohistochemistry, such as described in Example 1, to determine atwhat stage of development the PVCR protein is expressed in developingfish.

Based on the immunochemistry studies of the Aquatic PVCR structure,function and developmental expression, specific selection assays can bedesigned to identify fish, e.g., flounder, halibut or cod, species withaltered Aquatic PVCR function that can survive in fresh water, whilethose possessing normal PVCR function will die. These acute survivalassays can evaluate the overall effect of PVCR agonists and antagonistsidentified by e.g., the frog oocyte expression assay. These assays willtest the potency of various PVCR active compounds on improving orreducing survival of various fish or embryos. The ability to identify asingle individual fish with alterations in PVCR function andosmoregulation from many wild type fish possessing normalcharacteristics will permit the propagation of specific strains of fishthat exhibit specific salinity tolerance characteristics. Development oflarval forms of cod, halibut or flounder that survive in fresh water canthen be utilized in experiments to test whether new food sources couldbe used in their rearing.

Successful development of these goals would then permit these species tobe raised initially in protected fresh water hatcheries and latertransferred to marine conditions similar to those presently utilized foraquaculture of salmon.

Also encompassed by the present invention are methods of modulating theactivation of the Aquatic PVCR by altering the DNA encoding the AquaticPVCR, and thus, altering the subsequent expression of Aquatic PVCRprotein in various tissues. For example, anti-sense nucleic acidsequences (either DNA or RNA) can be introduced into e.g., epithelialcells in fish kidney, where the anti-sense sequence binds to the AquaticPVCR gene and inhibits, or substantially decreases its transcriptioninto MRNA. Alternatively, the anti-sense sequence can bind to theAquatic PVCR MRNA and inhibit, or substantially decrease, itstranslation into amino acid sequence.

Alternatively, a mutated or chimeric Aquatic PVCR gene construct (e.g.,a mutated or chimeric SEQ ID NO: 1) can be inserted into, e.g. fisheggs, to produce new marine strains with enhanced, or decreased, AquaticPVCR protein activity. The anti-sense sequence or gene construct isintroduced into the cells using techniques well-known to those of skillin the art. Such techniques are described in Hew, C. L., et al., Mol.Aguatic Biol. Biotech., 1:380717 (1992) and Du, S. J., et al.,Biotechnology, 10:176–181 (1992), the teachings of which areincorporated herein by reference.

Based on the work described herein, new methodologies that will regulatethe adaptation of fish, particularly flounder, halibut and cod, toenvironments of varying salinities are now available. For example,methods are now available to adapt developing forms of flounder, halibutor cod to fresh water environments. Rearing of these species in freshwater will allow for new approaches to the problems of feeding andsuccessful rearing of larval forms of these fish species. Methods arealso now available for selection and propagation of new strains of fish(e.g., flounder, halibut and cod) that will possess alterations in theirsalinity tolerance such that they can be raised in fresh water, thentransferred to seawater. This approach has many advantages since it willboth diversify the aquaculture industry and make use of existinghatcheries and facilities to produce flounder, cod or halibut as well assalmon.

The present invention is illustrated by the following Examples, whichare not intended to be limited in any way.

EXAMPLE 1 Immunohistochemistry of the PVCR Protein Present in AquaticSpecies Epithelial Cells

Tissues from fish were fixed by perfusion with 2% paraformaldehyde inappropriate Ringers solution corresponding to the osmolality of the fishafter anesthesitizing the animal with MS-222. Samples of tissues werethen obtained by dissection, fixed by immersion in 2% paraformaldehyde,washing in Ringers then frozen in an embedding compound, e.g., O.C.T.™Miles, Inc. Elkahart, Ind., using methylbutane cooled with liquidnitrogen. After cutting 4 μM tissue sections with a cryostat, individualsections were subjected to various staining protocols. Briefly, sectionsmounted on glass slides were: 1) blocked with serum obtained from thespecies of fish, 2) incubated with rabbit anti-CaR antiserum and 3)washed and incubated with peroxidase conjugated affinity purified goatantirabbit antiserum. The locations of the bound peroxidase conjugatedgoat antirabbit antiserum was visualized by development of a rosecolored aminoethylcarbazole reaction product. Individual sections weremounted, viewed and photographed by standard light microscopytechniques. The anti-CaR antiserum used to detect fish PVCR protein wasraised in rabbits using a 23-mer peptide corresponding to amino acidsnumbers 214–236 localized in the extracellular domain of the RaKCaRprotein.

In both species of elasmobranchs studied, (dogfish shark, SquatusAcanthias and little skate, Raja Erinacea), PVCR protein was localizedto the apical membranes of selected epithelial cells. The distributionof PVCR in elasmobranch tissue is shown in FIGS. 1A–F. Heavy blackcoloring is displayed where anti-CaR antibody binding is presentconsistently in areas of tissues designated by arrowheads. FIG. 1A:Kidney-CaR expression is present on apical membranes of epithelial cellsof late distal tubule (LDT) and collecting duct (CD). FIG. 1B: Gill PVCRexpression is localized to epithelial cells of gill arcades. FIG. 1C:Brain PVCR expression is localized to distinct groups of neurons in thebrain. FIG. 1D: Rectal gland PVCR expression is localized to apicalmembranes of cells lining the ducts of the rectal gland. FIG. 1E:Intestine PVCR expression is localized to the apical membranes ofepithelial cells lining the lumens of the intestine. FIG. 1F: Ovary PVCRexpression is present in both oocytes and surrounding follicular cells.

FIGS. 2A–F show the distribution of PVCR in the flounder(Pseudopleuronectes americanus) and in the fresh water trout(Onchorynchus Nerka). FIGS. 2A–F display heavy black coloring whereanti-CaR antibody binding is present consistently in areas of tissuesdesignated by arrowheads. FIG. 2A: Kidney-CaR expression is present onapical membranes of epithelial cells of large tubules (LT) andcollecting ducts (CD). FIG. 2B: Gill PVCR expression is localized toepithelial cells of gill arcades. FIG. 2C: Brain PVCR expression islocalized to distinct groups of neurons in the brain. FIG. 2D: Urinarybladder PVCR expression is localized to apical membranes of cells liningthe urinary bladder. FIG. 2E: Intestine PVCR expression is localized tothe apical membranes of epithelial cells lining the lumens of theintestine. FIG. 2F: Ovary PVCR expression is present in both oocytes andsurrounding follicular cells.

EXAMPLE 2 RNA Blotting Analyses of Winter Flounder Tissue

Five microgram samples of poly A+ RNA prepared from various winterflounder tissues including muscle (lane 1), heart (lane 2), testis (lane3) and urinary bladder (lane 4) were subjected to RNA blotting analyses(FIGS. 3A and B).

As shown in FIG. 3A, a single filter was first hybridized using a³²P-labeled ECO R1/XHO 15′ fragment of rat kidney PVCR cDNA (Brown, E.M., et al., Nature, 366:575 (1993)), washed at reduced stringency(1×SSC, 0.1% SDS, 50° C.) and exposed for 10 days to autoradiography.

As shown in FIG. 3B, the same filter shown in FIG. 3A after strippingand hybridization with a ³²P-labeled full length 3.8 kb TSC cDNA thatwas washed at 0.5×SSC, 0.1% SDS at 65° C. and subjected to a 1 hourautoradiogram exposure. Data shown representative of a total of fiveseparate experiments.

These data demonstrate the presence of a 4.4 kb homolog of the mammalianCaR present in poly A+ RNA from urinary bladder together with abundant3.8 kb thiazidesensitive NaCl contransporter transcript, and suggest noPVCR transcripts are present in other tissues including muscle, heart ortestis.

EXAMPLE 3 Molecular Cloning of Shark Kidney Calcuim Receptor RelatedProtein (SKCaR-RP)

A shark λZAP cDNA library was manufactured using standard commerciallyavailable reagents with cDNA synthesized from poly A+ RNA isolated fromshark kidney tissue as described and published in Siner et al. Am. J.Physiol. 270:C372–C381, 1996. The shark cDNA library was plated andresulting phage plaques screened using a ³²plabeled full length ratkidney CaR (RaKCaR) cDNA probe under intermediate stringency conditions(0.5×SSC, 0.1% SDS, 50° C.). Individual positive plaques were identifiedby autoradiography, isolated and rescued using phagemid infections totransfer CDNA to KS Bluescript vector. The complete nucleotide sequence,FIGS. 4A–E, (SEQ ID NO: 1) of the 4.1 kb shark kidney PVCR relatedprotein (SKCaR-RP) clone was obtained using commercially availableautomated sequencing service that performs nucleotide sequencing usingthe dideoxy chain termination technique. The deduced amino acid sequence(SEQ ID NO: 2) is shown in FIGS. 5A–E. Northern analyses were performedas described in Siner et. al. Am. J. Physiol. 270:C372–C381, 1996. TheSKCAR-RP nucleotide sequence was compared to others CaRs usingcommercially available nucleotide and protein database servicesincluding GENBANK and SWISS PIR.

Polymerase chain reaction (PCR) amplification of selected cDNA sequencessynthesized by reverse transcriptase (RT) were performed using acommercially available RT-PCR kit from Promega Biotech, Madison, Wis.Selective amplification of a conserved region of CaRs (nts 597–981 ofRaKCaR cDNA) results in 384 nt cDNA, as shown in FIG. 7. This amplified384 bp was then ligated into the TA cloning vector (Promega Biotech,Madison, Wis.) that was then transformed into competent DH5a E. colicells using standard techniques. After purification of plasmid DNA usingstandard techniques the 384 nt cDNA was sequenced as described above.

EXAMPLE 4 PVCR Epression in Tissue of Fundulus Herteroclitus

To determine if PVCR expression was modulated by adaptation of Fundulusto either fresh or salt water, killifish collected in an estuary werefirst fresh or salt water adapted for an interval of 18 days (chronicadaptation). Selected individuals from each group were then adapted tothe corresponding salinity (fresh to salt; salt to fresh) for aninterval of 7 days (acute adaptation).

Results are shown in FIG. 8. A blot containing RNA (40 ug/lane) preparedfrom control Xenopus kidney (lane 1) or Fundulus heart (containingultimobranchial tissue) (lanes 2, 5), kidney (lanes 3, 6) and gill(lanes 4, 7) was probed with a 32p-labeled Xenopus PVCR cDNA, washed(0.01 ×SSC, 650C) and autoradiographed.

As shown in FIG. 8, as compared to control MRNA, (lane 1) steady statelevels of PVCR MRNA are larger in tissues from seawater adapted fish(lanes 5–7) versus those in fresh water (lanes 2–4).

Fundulus fish were either chronically (FIGS. 9A and 9B) or acutely(FIGS. 9C and 9D) adapted to salt water (FIGS. 9A and 9C) or fresh water(FIGS. 9B and 9D). The presence of PVCR in kidney tubules was determinedby immunocytochemistry. Chronic adaptation to salt water (9A) resultedin increased PVCR expression in kidney tubules as compared to thatpresent in fresh (9B). Kidney tubule PVCR expression in salt water fishwas diminished by acute adaptation to fresh water (9C). In contrast,kidney tubule PVCR expression in fresh water fish was increased afteracute adaptation to salt water (9D).

EXAMPLE 5 Assay for PVCR Agonists and Antagonists Using the FlounderUrinary Bladder

To provide further evidence linking Aquatic PVCRs to fishosmoregulation, isolated urinary bladder of winter founder was used toinvestigate whether PVCRs modulate epithelial cell ion transport.Previous work has demonstrated that the flounder urinary bladder isimportant in osmoregulation since it allows recovery of both NaCl andwater via a thiazide-sensitive NaCl contransport process that has beenfirst generated by the kidney proximal tubule. Water reabsorption fromthe urine stored in urinary bladder allows for the concentrations ofboth Mg²⁺ and Ca²⁺ to increase to values as high as 84 mM and 7 mMrespectively in marine founders (Elger, E. B., et al., J. Como.Physiol., B157:21 (1987)). 5 Net apical to basolateral water flux (Jv)was measured gravimetrically in 10 minute intervals using individualurinary bladder excised from winter flounder. Briefly, isolated bladderswere suspended in a liquid solution (typically a physiologicallycompatible solution) as described in (Renfro, L. J. Am. J. Physiol.228:52–61, 1975) the teachings of which are hereby incorporated byreference. The weight of the bladder was measured before and after theexperimental period, wherein the experimental period comprised theperiod of time that the isolated bladder was exposed to test compound.The compound to be tested (e.g., test compound) was added to bothserosal and mucosal solutions. The bladders were dried and weighted asdescribed in Renfro et al. The difference in bladder weight prior to andafter exposure to test compound is an indication of water reabsorptionby the bladder.

Quantification of water reabsorption (Jv) by isolated bladders using themethod of Renfro et al. showed that Jv was significantly (p<0.05)inhibited by addition of 100 AM hydrochlorothiazide (86±2%) consistentwith the role of the thiazide sensitive NaCl contransporter in thisprocess. Urinary bladder iv was also significantly inhibited by PVCRagonists including 100 μM Gd³⁺(75±5%) and 200 μM neomycin (52±4%).(Control Jv values (130±28 μl/gm/hr.) were obtained from animals inSeptember–October and are approximately 21% of the Jv reported by Renfroet al. These differences likely reflect seasonal variations in urinarybladder transport.) The half maximal inhibitory concentration forurinary bladder Jv (IC₅₀) for Gd³⁺ (15 μM) was similar to that reportedfor mammalian CaRs, while the IC₅₀ for neomycin (150 μM) wasapproximately 3 times larger as compared to mammalian CaRs (50 μM). Thisinhibitory effect of PVCR agonists on Jv was fully reversible.Activation of apical PVCRS by high concentrations of Mg²⁺ and Ca²⁺resulting from NaCl-mediated water reabsorption from bladder urine wouldprovide for optimal recovery of water by the urinary bladder. Thismechanism would permit water reabsorption to proceed until divalentcation concentrations approach levels that promote crystal formation.This overall process is similar to that described for mammalian CaRs inthe rat and human IMCD. Additional aspects of these mammalian andteleost renal epithelia may also share other similarities since teleosturinary bladder is both an anatomical and functional homolog of themammalian mesonephric kidney.

EXAMPLE 6 Expression/Activation Studies of SKCaR in Human EmbryonicKidney (HEK) Cells

The following studies show the following:

-   -   1. SKCaR nucleic acid sequence (SEQ ID NO.: 1) encodes a        functional ion receptor that is sensitive to both Mg2+ and Ca2+        as well as alterations in NaCl.    -   2. SKCaR's (SEQ ID NO.: 2) sensitivity to Ca2+, Mg2+ and NaCl        occur in the range that is found in marine environments and is        consistent with SKCaRs role as a salinity sensor.    -   3. SKCaR's (SEQ ID NO.: 2) sensitivity to Mg2+ is further        modulated by Ca2+ such that SKCaR is capable to sensing various        combinations of divalent and monovalent cations in seawater and        freshwater. These data can be used to design novel electrolyte        solutions to maintain fish in salinities different from those        present in their natural environment.

SKCaR cDNA (SEQ ID NO.: 1) was ligated into the mammalian expressionvector PCDNA II and transfected into HEK cells using standardtechniques. The presence of SKCaR protein (SEQ ID NO.: 2) in transfectedcells was verified by western blotting. Activation of SKCaR (SEQ ID NO.:2) by extracellular Ca2+, Mg2+ or NaCl was quantified using a wellcharacterized FURA 2 based assay where increases in intracellular Ca2+produced by SKCaR activation are detected using methodology publishedpreviously the Dr. E. Brown's laboratory (Bai, M., S. Quinn, S. Trvedi,O. Kifor, S. H. S. Pearce, M. R. Pollack, K. Krapcho, S. C. Hebert andE. M. Brown. Expression and characterization of inactivating andactivating mutations in the human Ca2+-sensing receptor. J. Biol. Chem.,32:19537–19545 (1996)) and expressed as % normalized intracellularcalcium response to receptor activation.

SKCaR (SEQ ID NO.: 2) is a functional extracellular Ca2+ sensor whereits sensitivity is modulated by alterations in extracellular NaClconcentrations. As shown in FIG. 10, SKCaR (SEQ ID NO.: 2) is activatedby increasing concentrations of extracellular Ca2+ where half maximalactivation of SKCaR (SEQ ID NO.: 2) ranges between 1–15 mM depending onthe extracellular concentration of NaCl. These are the exact ranges ofCa2+(1–10 mM present in marine estuarian areas). Note that increasingconcentrations of NaCl reduce the sensitivity of SKCaR (SEQ ID NO.: 2)to Ca2+(see Panel B). This alteration in SKCaR (SEQ ID NO.: 2)sensitivity to Ca2+ was not observed after addition of an amount ofsucrose sufficient to alter the osmolality of the extracellular medium.This control experiment shows it is not alterations in cell osmolalityeffecting the changes observed.

The half maximal activation (EC50) by Ca2+ for SKCaR (SEQ ID NO.: 2) isreduced in increased concentrations of extracellular NaCl. See FIG. 11.The EC50 for data shown on FIG. 10 is displayed as a function ofincreasing extracellular NaCl concentrations. Note the EC50 for Ca2+increases from less than 5 mM to approximately 18 mM as extracellularNaCl concentrations increase from 50 mM to 550 mM.

SKCaR (SEQ ID NO.: 2) is a functional extracellular Mg2+ sensor whereits sensitivity is modulated by alterations in extracellular NaClconcentrations. As shown in FIG. 12, SKCaR (SEQ ID NO.: 2) is activatedin the range of 5–40 mM extracellular Mg2 +and is modulated in a mannersimilar to that shown in FIGS. 10 and 11 by increasing concentrations ofextracellular NaCl. Similarly, this alteration in SKCaR (SEQ ID NO.: 2)sensitivity to Ca2+ was not observed after addition of an amount ofsucrose sufficient to alter the osmolality of the extracellular medium.

The half maximal activation (EC50) by Mg2+ for SKCaR (SEQ ID NO.: 2) isreduced in increased concentrations of extracellular NaCl. See FIG. 13.The EC50 for data shown on FIG. 12 is displayed as a function ofincreasing extracellular NaCl concentrations. Note the EC50 for Mg2+increases from less than 20 mM to approximately 80 mM as extracellularNaCl concentrations increase from 5 OmM to 550 mM.

Addition of 3 mM Ca2+ alters the sensitivity of SKCaR (SEQ ID NO.: 2) toMg2+ and NaCl. See FIG. 14. The EC50 for Mg2+ of SKCaR (SEQ ID NO.: 2)is modulated by increasing concentrations of NaCl as shown both in thisFIG. 14 and in FIG. 13. Addition of 3 mM Ca2+ to the extracellularsolution alters the sensitivity characteristics of SKCaR (SEQ ID NO.: 2)as shown. Note the 3 mM Ca2+ increases the sensitivity of SKCaR (SEQ IDNO.: 2) to Mg2+ as a function of extracellular NaCl concentrations.

EXAMPLE 7 Demonstration of the Presence of a Functional PVCR in UrinaryBladder of Winter Flunder.

Quantification of water reabsorption (J_(v)) in isolated bladders (ref.Renfro, J. L. Water and ion transport by the urinary bladder of theteleost Pseudopleuronectes americanus. Am. J. Physiol. 228:52–61 (1975)showed that control Jv (130±28 μl/gm/hr; n=14) was significantly(p<0.05) inhibited (86±2%) by addition of 100 μM hydrochlorothiazide(18±7 μl/gm/hr; n=6) consistent with the role of the thiazide-sensitiveNaCl cotransporter in this process. Urinary bladder J_(v) was alsoinhibited significantly by CaR agonists including 100 μM Gd³⁺ (75±5%inhibition; 32±18 μl/gm/hr; n=5) and 200 μM neomycin (52±4% inhibition;63±10 μl/gm/hr; n=5). The half maximal inhibitory concentration forurinary bladder J_(v), (IC₅₀) for Gd³⁺ (15±3 μM; n=6) was similar tothat reported for mammalian CaRs (See Brown, E. M., G. Gamba, D.Riccardi, D. Lombardi, R. Butters, O. Kifor, A. Sun, M. Hediger, J.Lytton and S. C. Hebert. Cloning and characterization of anextracellular Ca ²⁺ sensing receptor from bovine parathyroid. Nature366:575–580 (1993) while the neomycin IC₅₀(150±24 μM; n=6) wasapproximately 2–3 fold higher than for mammalian CaRs (60–70 μM) (Brown,E. M., G. E. -H. Fuleihan, C. J. Chen and O. Kifor. A comparison of theeffects of divalent and trivalent cations on parathyroid hormonerelease) 3′5′-cyclic-adenosine monophosphate accumulation and the levelsof inositol phosphates in bovine parathyroid cells. Endocrinol.127:1064–1071 (1990).

The maximal inhibitory effect for both CaR agonists on J_(v), was fullyreversible as shown in FIG. 15.

Response of a single isolated urinary bladder of winter flounder afterexposure of its apical membrane to various CaR agonists andhydrochlorothiazide is shown in FIG. 15. Water transport (Jv) wasmeasured in a single isolated urinary bladder after sequential exposuresto 300 μM Gd3+, 100 mM thiazide and 100 mM Mg2+. Note that full recoveryof water transport occurred after exposure to each of these agents. Thisdata validates of the use of isolated urinary bladder as a screeningassay.

EXAMPLE 8 Immunochemistry showing that PVCR Exists in Olfactory Organs

Additional immunocytochemistry experiments were performed using antibody1169 (the antibody raised against the 23-mer peptide described herein)to localize SKCaR protein where it is present on the apical membrane ofthe lamellae of the olfactory organ epithelia of the dogfish shark(Squalus ancanthias). These data suggest that elasmobranchs possess theability to “smell” salinity gradients in the marine environments.Furthermore, from this location SKCaR may interact with other odorantreceptors that are also 7 transmembrane GTP binding protein receptors.

FIG. 31A shows the immunocytochemistry of the lamellae of the olfactoryorgan epithelia of the dogfish shark (Squalus ancanthias) using antisera1169. Note the brown reaction product indicating specific 1169 antibodybinding to the apical membrane of olfactory organ epithelial cells. FIG.31B also is a photograph that shows lamellae that is not subject toantisera 1169, the control.

EXAMPLE 9 PVCRs Isolated in Various Aquatic Species

The PVCR has been isolated in several species including winter flounder(sole), summer flounder (fluke) and lumpfish (source of caviar). ThePVCR has also been isolated in swordfish and lamprey. In addition, 2sequences distinct from SKCaR-I have been obtained from shark indicatingthere are multiple polyvalent cation sensing receptors in a singlespecies of fish.

Sequences of mammalian CaRs together with the nucleotide sequence ofSKCaR (SEQ. ID NO: 1 and SEQ ID NO: 2) were used to design degenerateoligonucleotide primers to highly conserved regions in the extracellulardomain of polyvalent cation receptor proteins using standardmethodologies (See G M Preston, Polymerase chain reaction withdegenerate oligonucleotide primers to clone gene family members, Methodsin Mol. Biol. Vol. 58 Edited by A. Harwood, Humana Press, pages 303–312,1993). Using these primers, cDNA or genomic DNA from various fishspecies representing important commercial products are amplified usingstandard PCR methodology. Amplified bands are then purified by agarosegel electrophoresis and ligated into appropriate plasmid vector that istransformed into a bacterial strain. After growth in liquid media,vectors and inserts are purified using standard techniques, analyzed byrestriction enzyme analysis and sequenced where appropriate. Using thismethodology, a total of 5 nucleotide sequences from 4 fish species wereamplified.

Two additional nucleotide sequences were isolated from the Dogfish shark(Squalus ancanthias), same species as SKCaR-I (SEQ ID NO:2). Twonucleotide sequences, SEQ ID NO: 3 (FIGS. 16A–B) and SEQ ID NO: 5 (FIG.19), were isolated from genomic SEQ ID NO: 3 or cDNA obtained from sharkrectal gland (SEQ ID NO: 5). Both SEQ ID NOs: 3 and 5 are unique ascompared to corresponding regions of the nucleotide sequence of SKCaR-I(SEQ ID NO: 1). SEQ ID NOs: 4 and 6 (FIGS. 17 and 20, respectively)represent the corresponding amino acids of putative open reading framesof SEQ ID NOs: 3 and 5. Thus, these 2 sequences represent at least 1(different fragments of a single other gene) or possibly 2 calciumpolyvalent cation sensing receptor proteins distinct from the SKCaR-I.FIG. 18 and FIG. 21 show the nucleotide sequences for SEQ ID NOs: 3 and5, respectively, and the corresponding deduced amino acid sequences (SEQID NOs: 4 and 6, respectively).

SEQ ID NO: 3 is composed of 784 nucleotides (nt) containing an openreading frame coding for 261 amino acids. SEQ ID NO: 3 is similar, butnot identical to the corresponding sequence in the extracellular domainof SKCaR I (SEQ ID NOs: 1 and 2) from nt. 1087–1836.

SEQ ID NO: 4 is composed of 261 Amino acids corresponding to theputative open reading for SEQ ID NO: 3.

SEQ ID NO: 5 is composed of 598 nucleotides (nt) containing an openreading frame coding for 198 amino acids and was obtained usingoligonucleotide primers different from those used for SEQ ID NO: 3. SEQID NO: 5 is similar, but not identical to the corresponding sequence inthe extracellular domain of SKCaR I (SEQ ID NOs: 1 and 2) from nt.2279–2934.

SEQ ID NO: 6 comprises 198 Amino acids corresponding to the putativeopen reading for SEQ ID NO: 4.

Winter Flounder (Pleuronectes americanus) marine flatfish species wasalso isolated using the techniques described herein. SEQ ID NO: 7 wasobtained from cDNA prepared from urinary bladder where functional datashow presence of PVCR protein. SEQ ID NO: 8 corresponds to amino acidsin the putative open reading frame of SEQ ID NO: 7.

SEQ ID NO: 7 is composed of 594 nucleotides (nt) containing an openreading frame coding for 197 amino acids. SEQ ID NO: 7 is homologous tothe corresponding sequence in the extracellular domain of SKCaR I (SEQID NOs: 1 and 2) from nt. 2279–2937.

SEQ ID NO: 8 comprises the 197 Amino acids corresponding to the putativeopen reading frame of SEQ ID NO: 7.

Summer Flounder (Paralichthus dentalus) is another marine flatfishspecies that was isolated using methods, as described herein. SEQ ID NO:9 was obtained from cDNA prepared from urinary bladder that is similarin function to the urinary bladder of winter flounder. SEQ ID NO: 10contains amino acid corresponding to the putative open reading frame ofSEQ ID NO: 9.

SEQ ID NO: 9 is composed of 475 nucleotides (nt) containing an openreading frame coding for 157 amino acids. SEQ ID NO: 9 is homologous tothe corresponding sequence in the extracellular domain of SKCaR I (SEQID NOs: 1 and 2) from nt. 2279–2934.

SEQ ID NO: 10 has 157 Amino acids corresponding to the open readingframe for SEQ ID NO: 9.

Lumpfish (Cyclopterus rumpus) is an arctic marine fish that wasisolated. Lumpfish is the sole source of lumpfish caviar. SEQ ID NO: 11was obtained from cDNA prepared from the urinary bladder of lumpfish.SEQ ID NO: 12 is the corresponding amino acid sequence of the putative435 amino acid open reading frame of SEQ ID NO: 11.

SEQ ID NO: 11 is composed of 1308 nts. that are homologous to thecorresponding sequence in the extracellular domain of SKCaR I (SEQ IDNO: 1 and 2) from nt 1087–2441.

SEQ ID NO: 12 comprises the 435 Amino acids corresponding to theputative open reading frame for SEQ ID NO: 11.

Sequences derived from Primer sequences for PCR of PVCR clones: thefollowing SEQ ID NOs: dSK-F1 5′-GCI GCT GAY GAY GAY TAY GG-3′ (SEQ IDNO.: 15) 3, 11 dSK-R2 5′-CCA IGC YTC IAG YTT YTT DAT RTC-3″ (SEQ ID NO.:16) 3 dSK-F3 5′-TGT CKT GGA CGG AGC CCT TYG GRA TCG C-3′ (SEQ ID NO.:17) 5, 7, 9 dSK-R3 5′-ATA GGC KGG RAT GAA RGA KAT CCA RAC RAT GAA G-3′(SEQ ID NO.: 18) 7 dSK-R4 5′-GGC KGG RAT GAA RGA KAT CCA RAC RAT GAAG-3′ (SEQ ID NO.: 19) 5, 9, 11 I = deoxyinosine, N = A + C + T + G, R =A + G, Y = C + T, M = A + C, K = T + G, S = C + G, W = A + T, H = A +T + C, B = T + C + G, D = A + T + G, V = A + C + G

EXAMPLE 10 Altering the Body Composition of Fish and Defining SalinityLimits

Winter and Summer Flounder can be grown and maintained in recyclingwater systems. Groups of both winter (Pleuronectes americanus) andsummer (Paralichthus dentalus) flounder were maintained in multiplemodular recycling water system units that are composed of a single 1meter fish tank maintained by a 1 meter biofilter tank located directlyabove it. The upper tank of each unit contains 168 sq. ft. of biofiltersurface area that will support a maximum of 31 lbs of flounder, whilemaintaining optimal water purity and oxygenation conditions. Each unitis equipped with its own pump and temperature regulator apparatus. Boththe temperature and photoperiod of each unit can be independentlyregulated using black plastic curtains that partition each tank off fromits neighbor. The inventors have a total of 12 independent modular unitsthat permit 3 experiments each with 4 variables to be performedsimultaneously. Using this experimental system, the following data havebeen obtained.

Salinity survival limits for winter and summer flounder with a constantratio of divalent and monovalent ions were determined. The survivallimit of both winter and summer flounder in waters of salinities greaterthan normal seawater (10 mM Ca2+, 50 mM Mg2+ and 450 mM NaCl) is watercontaining twice (20 mM Ca2+, 50 mM Mg2+ and 900 mM NaCI) the normalconcentrations of ions present in normal seawater. In contrast, thesurvival limit of both winter and summer flounder in waters of salinityless than normal seawater is 10% seawater (1 mM Ca2+, 5 mM Mg2+ and 45mM NaCl).

Flounder grown and/or maintained in low and hypersalinities possessdifferent fat contents and taste as compared to flounder maintained innormal sea water. Use of a fully recycling water system permits growthof flounder at vastly different salinities. Groups of flounder (n=10)were adapted over a 15 day interval and maintained at either lowsalinity (LS) (e.g., at 10% normal seawater), normal seawater (NS) orhypersalinity (HS) (e.g., 2× seawater) for intervals of 3 months, underotherwise identical conditions. Survival among the 3 groups werecomparable (all greater than 80%) and there were no differences in theelectrolyte content of their respective sera. Analyses of fillet musclefrom summer flounder for total fat, protein and moisture content areshown on Table I.

TABLE I Comparison of Total Fat, Protein and Moisture Content of Musclefrom Flounders Grown at Differing Water Salinities for 3 months. Allvalues an average of 4 individual fish. Salinity 10% Seawater NormalSeawater 2× Seawater % Total Fat  3.36 ± 0.43*  2.59 ± 0.31*  1.98 ±0.66* % Total Protein 19.6 ± 0.23 19.9 ± 0.42 18.99 ± 0.34  % Moisture74.7 ± 2.1  75.1 ± 1.8  73.8 ± 2.5  *Values significantly different fromeach other (p < 0.05).

Muscle from low salinity flounder contains approximately 30% higher fatcontent as compared to flounder maintained in normal seawater andapproximately 70% greater fat content when compared to floundermaintained in 2× seawater (e.g., the fat of a flounder maintained innormal salinity is 40% greater than flounder maintained in twiceseawater). These differences appear selective because no significantdifferences were observed in either muscle protein or moisture content.

Furthermore, fillets were sampled in a blinded protocol where tasters(n=6) were offered either raw or cooked fillets without knowledge ofsalinity conditions. Tasters could distinguish little difference betweenthe taste of fillets of individual fish from each specific salinitygroup. However, when asked to compare fillets from flounder grown atdiffering salinities, a majority (⅚) clearly distinguished a tastedifference between fillets from fish maintained at 10% salinitydescribing them as “sweet and buttery tasting with a soft consistency”as compared to fillets from fish maintained at either normal seawater or2× seawater that were described as “wild and fishy tasting with a firmerconsistency. These data provides evidence that “finishing” growth ofwinter flounder at different water salinities can be used to alter thetaste and fat content of the resulting fillets in summer and winterflounder.

Groups of tagged hatchery raised summer flounder obtained from identicalbroodstock were exposed to either 10% seawater or 2× seawater for aninterval of 3 months under conditions identical to that described above.There were no significant differences in either length or width in fishmaintained 10% seawater or 2× seawater. However, there was a significantdifference in the weights of the respective fish where 10% seawater fishweighted 80±14% (n=10) more than summer flounder maintained in 2×seawater. Moreover, the summer flounder maintained in 10% seawater werenearly twice (2.1±0.4 times n=6) as thick as compared to fish maintainedin 2× seawater. These data show that flounder maintained at differentwater salinities exhibit significant differences in the thickness oftheir fillets. Thus, flounder could be “finished” using water ofdiffering compositions to alter the thickness of their fillets.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

1. An isolated polypeptide molecule having at least about 80% identitywith a) SEQ ID NO: 2; or b) an amino acid sequence encoded by thenucleic acid sequence of SEQ ID NO: 1; wherein the isolated polypeptidemolecule allows fish to sense Ca²⁺, Mg²⁺, or Na⁺ ion concentrations. 2.An isolated polypeptide molecule having at least about 90% identity witha) SEQ ID NO: 2; or b) an amino acid sequence encoded by the nucleicacid sequence of SEQ ID NO: 1; wherein the isolated polypeptide moleculeallows fish to sense Ca²⁺, Mg²⁺, or Na⁺ ion concentrations.
 3. Anisolated polypeptide molecule having at least about 80% identity with a)SEQ ID NO: 2; or b) an amino acid sequence encoded by the nucleic acidsequence of SEQ ID NO: 1; wherein the isolated polypeptide moleculeassists fish in adapting to changing Ca²⁺, Mg²⁺, or Na⁺ ionconcentrations by altering water intake, water absorption or urineoutput.
 4. An isolated polypeptide molecule having at least about 90%identity with a) SEQ ID NO: 2; or b) an amino acid sequence encoded bythe nucleic acid sequence of SEQ ID NO: 1; wherein the isolatedpolypeptide molecule assists fish in adapting to changing Ca²⁺, Mg²⁺, orNa⁺ ion concentrations by altering water intake, water absorption orurine output.
 5. An isolated polypeptide molecule having at least about80% identity with a) SEQ ID NO: 2; or b) an amino acid sequence encodedby the nucleic acid sequence of SEQ ID NO: 1; wherein the isolatedpolypeptide molecule allows a fish to modulate the percentage of totalfat, protein and moisture of muscle and allows fish to sense or adapt toCa²⁺, Mg²⁺, or Na⁺ ion concentrations.
 6. An isolated polypeptidemolecule having at least about 90% identity with a) SEQ ID NO: 2; or b)an amino acid sequence encoded by the nucleic acid sequence of SEQ LIDNO: 1; wherein the isolated polypeptide molecule allows a fish tomodulate the percentage of total fat, protein and moisture of muscle andallows fish to sense or adapt to Ca²⁺, Mg²⁺, or Na⁺ ion concentrations.7. An isolated polypeptide molecule having an amino acid sequence thatcomprises: a) SEQ ID NO: 2; or b) an amino acid sequence encoded by thenucleic acid sequence of SEQ ID NO: 1 wherein the isolated polvpeptidemolecule allows fish to sense or adapt to Ca²⁺, Mg²⁺, or Na⁺ ionconcentrations.
 8. An isolated polypeptide encoded by a nucleic acidsequence of a clone deposited under ATCC No.: 209602, wherein theisolated polypeptide molecule allows fish to sense or adapt to Ca²⁺,Mg²⁺, or Na⁺ ion concentrations.